The transposon Tip100 from the common morning glory is an ...

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Abstract The mutable ¯aked or a¯aked (af) line of the common morning glory (Ipomoea purpurea) displays white flowers with colored flakes, and the af mutation ...
Mol Genet Genomics (2002) 266: 732±739 DOI 10.1007/s00438-001-0603-z

O R I GI N A L P A P E R

N. Ishikawa á Y. Johzuka-Hisatomi á K. Sugita H. Ebinuma á S. Iida

The transposon Tip100 from the common morning glory is an autonomous element that can transpose in tobacco plants Received: 18 June 2001 / Accepted: 4 October 2001 / Published online: 8 November 2001 Ó Springer-Verlag 2001

Abstract The mutable ¯aked or a¯aked (af) line of the common morning glory (Ipomoea purpurea) displays white ¯owers with colored ¯akes, and the af mutation is caused by the insertion of a transposable element named Tip100 into the CHS-D gene for anthocyanin biosynthesis. The 3.9-kb Tip100 element belongs to the Ac/Ds family and contains an ORF encoding a polypeptide of 808 amino acids. The frequency and timing of ¯ower variegation vary in di€erent af lines, and a genetic element termed Modulator has been postulated to a€ect the variegation pattern. Since the pattern of ¯ower variegation is determined by the frequency and timing of excision of Tip100 from the CHS-D gene, we wished to determine whether Tip100 is an autonomous element that is itself capable of transposition in a heterologous host. To do this, we introduced the element into the genome of tobacco plants by Agrobacterium-mediated transformation. The intact Tip100 element was able to excise from its original position in the chromosome and reinsert into new sites in the tobacco genome, whereas an internal deletion derivative was not. Based on these results, we conclude that Tip100 is an autonomous element. We also discuss the nature of the putative Modulator element a€ecting ¯ower and leaf variegation in various mutable lines of the morning glory.

Communicated by J. Schell N. Ishikawa á Y. Johzuka-Hisatomi á S. Iida (&) National Institute for Basic Biology, Myodaiji, Okazaki-shi, Aichi 444-8585, Japan E-mail: [email protected] Fax: +81-564-557685 N. Ishikawa á S. Iida The Graduate University for Advanced Studies, Okazaki-shi, Aichi 444-8585, Japan K. Sugita á H. Ebinuma Pulp and Paper Research Laboratory, Nippon Paper Industries Co Ltd., Kita-ku, Tokyo 114-0002, Japan

Keywords Ac/Ds family á Autonomous element á Flower and leaf variegation á Ipomoea purpurea á Transposable element Tip100

Introduction Transposable DNA elements that move by excision and reintegration can be classi®ed as autonomous or nonautonomous (Kunze et al. 1997). Autonomous elements carry both cis-acting elements required for transposition and the complete coding regions for trans-acting transposases, whereas non-autonomous elements, in which the transposase gene is defective or lacking, can be mobilized only when active transposases are supplied by an autonomous element elsewhere in the genome. Somatic excisions of these elements from genes involved in pigmentation often cause a variegated phenotype. The best studied mutation in the common morning glory (Ipomoea purpurea or Pharbitis purpurea) is a mutable allele of the A locus termed ¯aked, also called anthocyanin¯aked (a¯aked or af) (Barker 1917; Imai and Tabuchi 1935; Kasahara 1956; Epperson and Clegg 1987; Hisatomi et al. 1997; Habu et al. 1998; Iida et al. 1999; Durbin et al. 2001; Hoshino et al. 2001). In the homozygous state af/af, the mutable ¯aked lines display white ¯owers with colored ¯akes (Fig. 1A). The ¯ower variegation is thought to be caused by recurrent somatic reversion of the non-functional allele (white) to functional (pigmented). One of the characteristics of the mutable ¯aked allele is that the timing and frequency of ¯ower variegation vary in di€erent lines, and the variegation pattern is generally heritable (Kasahara 1956; Habu et al. 1998). We have shown that the mutable af allele has an insertion of the 3.9-kb transposable element Tip100 in the intron of the CHS-D gene encoding chalcone synthase for ¯ower pigmentation (Habu et al. 1998; Shiokawa et al. 2000; Hoshino et al. 2001). Tip100 in the CHS-D intron carries 11-bp terminal inverted repeats (TIRs) and is ¯anked by

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Fig. 1A, B Phenotypes of Ipomoea purpurea carrying the mutable af and yglm alleles for ¯ower and leaf variegation, respectively. The structure of the af allele with Tip100 inserted in the CHS-D gene is also shown. The ®lled boxes indicate the exon sequences of the CHS-D gene. The mutable yglm allele remains to be identi®ed. A KK/VR-40a (Modulator active). B KK/WR321 (Modulator inactive)

target-site duplications (TSDs) of 8 bp. It contains an ORF encoding a polypeptide of 808 amino acids which exhibits partial homology to the conserved regions of the transposase of the Ac/Ds family. These structural features are compatible with the notion that Tip100 may be an autonomous element belonging to the Ac/Ds family. Kasahara (1956) described a line of the common morning glory showing variegated leaf pigmentation, characterized by the appearance of dark green spots and sectors on a yellow-green background (Fig. 1A). This mutable allele was named yellow-green leafmutable, or yglm (previously described as y'). It was further noted that the timing and frequency of the variegated phenotype in leaves are also generally heritable and that a plant carrying both af and yglm tends to show a striking similarity in both timing and frequency of the variegations in ¯owers and leaves (Fig. 1). Based on these observations, Kasahara (1956) postulated that there must be another genetic element, termed Modulator (originally named Mutator), that acts on both the af and yglm alleles, and that the timing and frequency of variegation in ¯owers and leaves are determined by the heritable state of the Modulator. By crossing them with an active Modulator line, it is possible to distinguish a mutant lines bearing white ¯owers due to a stable mutation at the A locus from white-¯ower mutants carrying the af allele without an active Modulator. Subsequently, we found that lightly and heavily variegated ¯ower lines, as well as the white-¯ower line with inactive Modulator, all

carry a single Tip100 insertion at the same site in the CHS-D intron (Fig. 1; Habu et al. 1998). In contrast, the stable white-¯ower line with active Modulator has two insertions of Tip100 within the CHS-D intron. Excision of one of the two copies of Tip100 from the CHS-D gene in the latter stable white-¯ower line appears to be insucient to restore the CHS-D function, and probably both elements are rarely excised in the same tissue (Habu et al. 1998). The simplest explanation for these observations is that Tip100 and Modulator correspond to a non-autonomous and an autonomous element, respectively. Alternatively, Tip100 may itself be an autonomous element and Modulator an element which can control the transposition activity of Tip100. To determine whether Tip100 is an autonomous element, we introduced Tip100 and its internal deletion derivative dTip100 into tobacco (Nicotiana tabacum), and examined whether they are able to transpose in the transgenic tobacco plants. Our data strongly indicate that Tip100 carries all the functions necessary for its own transposition, and therefore represents the ®rst autonomous element to be characterized in the genus Ipomoea.

Materials and methods Plasmid vectors The Tip100 element ¯anked by 8-bp TSDs in the clone kV-Tip1004S7 (Habu et al. 1998) was ampli®ed by PCR and cloned into the PstI site of pHSG398 (TaKaRa Biomedicals) to yield pHSG 398::Tip100. The primers used for PCR ampli®cation were: Tip100L (5¢-GGCTGCAGCATACGTGCAGGGGCGGAGGCA C-3¢) and Tip100-R (5¢-GGCTGCAGCACGTATGCAGGGGCGGAG CCAGGATTA-3¢), which contain a PstI site (boldface), the 8-bp TSD sequence (italics) originating from the CHS-D intron, and the Tip100 terminal sequences (underlined). The internal 1.7-kb SpeI segment of Tip100 in pHSG398::Tip100 was deleted to yield pHSG398::dTip100. The plasmid pSUG1 is a derivative of pBI121 (Clontech) and carries a unique Sse8387I site at the SmaI site of pBI121. To construct pSUG1::Tip100 and pSUG1::dTip100 (Fig. 2), the PstI fragment containing either Tip100 or dTip100 was cloned into the Sse8387I site of pSUG1, thus separating the GUS gene from the 35S promoter. The BamHI-SacI fragment containing the GUS gene for b-glucuronidase in pBI121 was replaced by the BamHI-SacI fragment containing the hph gene for hygromycin B phosphotransferase and the CaMV 35S terminator from pCKR138 (Izawa et al. 1991) to yield pSUG2. To obtain pSUG2::Tip100, the PstI fragment containing Tip100 was cloned into the Sse8387I site of pSUG2. Transgenic plants Agrobacterium-mediated transformation of the plasmid vectors pSUG1::Tip100 and pSUG1::dTip100 into N. tabacum cv. SR1 plants was performed as previously described (Sugita et al. 1999). After co-cultivation of leaf segments with A. tumefaciens LBA4404 (Hoekema et al. 1983) containing the appropriate plasmid on solid hormone-free MS medium containing 50 mg/1 acetosyringone for 3 days, the explants were transferred to solid SIM medium (MS

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Fig. 2 Structures of the T-DNA regions in the plasmid vectors used. The ®lled arrowheads labeled L and R represent the left and the right borders of T-DNA, respectively. The open triangles and the thick horizontal arrows within the Tip100 boxes indicate TIRs and ORFs, respectively. The boxes marked PN and TN indicate the promoter and the terminator of the nopaline synthase gene, respectively, and the boxes labeled P35 and T35 represent the CaMV 35S promoter and the CaMV 35S terminator, respectively. The structural genes for Kmr, GUS and Hmr are indicated by NPTII, GUS and HPH, respectively. The thin arrows below the maps indicate the positions of primers used for PCR or IPCR ampli®cation. Note that both Tip100 and dTip100 on the pSUG1 derivatives are ¯anked by the 8-bp TSDs originating from the ¯aked allele of the CHS-D gene (Habu et al. 1998). Restriction sites are: Ba, BamHI; Ps, PstI; Sa, SacI; Sp, SpeI

CTGCCAGTTCAGT-3¢) were cloned into the pGEM T-easy plasmid (Promega) and subsequently sequenced with the primer GUS-2 (5¢-TCGCGATCCAGACTGAATGCCC-3¢). Excision of dTip100 promoted by Tip100 Agrobacterium-mediated transformation of pSUG2::Tip100 or pSUG2 into a transgenic plant (dTip1) carrying one copy of the NPTII gene was performed in the same way as the transformation of pSUG1::Tip100 described above, except that hygromycin (50 mg/l) was used instead of kanamycin (100 mg/l) for selection and the regeneration step on hormone-free MS medium was omitted. Hygromycin-resistant (Hmr) calli were used for further analyses. Reintegration of Tip100

medium containing 1 mg/l benzyladenine, 0.1 mg/l naphthalene acetic acid and 100 mg/l kanamycin) containing 500 mg/1 ticarcillin to prevent further bacterial growth. One month after infection with Agrobacterium, regenerated adventitious buds that were resistant to kanamycin (Kmr) were removed and transferred to hormone-free MS medium containing 100 mg/l kanamycin and 500 mg/l ticarcillin. To estimate the number of integrated T-DNA copies present in transgenic tobacco plants, we employed the inverse PCR (IPCR) procedure using the primers P1 (5¢-CGTTGCGGTTCTGTCAG TTCC-3¢) and P2 (5¢-TTGTCAAGACCGACCTGTCC-3¢) to determine the copy number of the NPTII gene for neomycin phosphotransferase (Fig. 2; Does et al. 1991). To examine the excision of Tip100 from the introduced GUS gene, we carried out a phenotypic assay for GUS expression in the leaves of transgenic tobacco plants using the GUS staining procedure previously described by Je€erson et al. (1987). To determine the footprint sequences generated by Tip100 excision (Fig. 2), the fragments produced by PCR ampli®cation with the primers FT-1 (5¢-ACAATCCCACTATCCTTCGC-3¢) and FT-2 (5¢-GGATAGT

To determine the sequences at the sites of reintegration of Tip100, we used IPCR ampli®cation using the primers TIR-1 (5¢-GGC CAA GCCGCCAAGGCCCCTATTGCCTAATAGGC-3¢) and TIR-2(5¢-CGTGACATGCAAGAGAGACGGTCTAAATT TTAGT-3¢), followed by nested PCR ampli®cation with the primers TIR-3 (5¢-GGGCCAAGGCCCCCTGCATATAATATGTGC-3¢) and TIR-4 (5¢-TTTCAGTATTCTGTCTTAAAT TGTGATT-3¢). The positions of these primers within Tip100 are shown in Fig. 2. Since Tip100 contains no KpnI site, the genomic DNAs extracted from leaves of the transgenic plants to be examined were digested with KpnI. After circularizing the KpnI fragments by self-ligation, DNA samples were treated with PstI to destroy the ampli®ed DNA fragments originating from the integrated pSUG1::Tip100 sequences (the initially integrated, non-transposed, Tip100 element is ¯anked by the PstI sequences used for construction of pSUG1::Tip100). After the ®rst PCR with the primers TIR-1 and TIR-2, the reaction mixture was diluted 100-fold and subjected to nested PCR ampli®cation with the primers TIR-3 and TIR-4. The ampli®ed fragments were cloned into pGEM T-easy and the TSD sequences were determined.

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Results and discussion A phenotypic assay for the excision of Tip100 from a GUS gene introduced into transgenic tobacco plants To examine whether Tip100 is an autonomous element, we introduced Tip100, inserted in the GUS gene, into tobacco plants, and examined whether it can excise from the reporter gene in the transgenic plants. For this test, we also employed the dTip100 element, in which the ORF of Tip100 is disrupted by deletion of the internal 1.7-kb SpeI fragment, as a control defective element (Fig. 2). Since the inserted Tip100 and dTip100 elements eciently prevent GUS expression from pSUG1::Tip100 and pSUG1::dTip100, respectively, the GUS gene will be activated if the elements are excised. Tip100 in pSUG1::Tip100 and dTip100 in pSUG1::dTip100 were introduced into tobacco by Agrobacterium-mediated transformation, and Kmr transgenic plants were obtained. Most of these primary transformants (T0) were found to contain only one copy of the integrated NPTII gene in their genomes; the rest carried two or three copies of the NPTII gene (see Table 1). Excision of Tip100 or dTip100 from the GUS gene was examined in leaves from these T0 plants by assaying for GUS activity. As Fig. 3A shows, the leaves of all twelve independent T0 plants with pSUG1::Tip100 examined displayed rare but clear and small blue, GUS-positive, patches when stained with X-Gluc (5-bromo-chloro3-indolyl-beta-D-glucuronide), indicating that Tip100 excision from the GUS gene occurred in the transgenic tobacco plants. We noticed that Tip100 excision tends to occur much more frequently in the plants at a younger stage, and no obvious GUS-positive blue spots are observed to arise in the same plants at later stages of development. Preliminary results suggested that the introduced Tip100 elements appear to be heavily methylated at later stages in the development of the transgenic plants. A comparable observation was reported for an autonomous Tam3 element belonging to the Ac/Ds family that was introduced into transgenic tobacco plants; stabilization appeared to be due to rapid methylation of the element (Martin et al. 1989). However, GUS-positive spots or sectors could be detected in the selfed T1 progeny derived from the T0 plants (Fig. 3B, C). None of the three transformants with pSUG1::dTip100 showed any GUS-positive spots at all (data not shown). These results indicate that Tip100 can be excised from the GUS gene in transgenic tobacco plants, whereas dTip100 cannot. This suggests that Tip100 is likely to be an autonomous element coding for an active transposase of 808 amino acids. A phenotypic assay for the Tip100-mediated excision of dTip100 from the GUS gene We asked whether dTip100, in which the transposase gene is non-functional due to an internal 1.7-kb deletion,

could be excised from the GUS gene when the intact Tip100 element was introduced into cells containing the defective element in the GUS gene integrated in the genome. To examine this possibility, we chose the T0 tobacco plant dTip1, which appears to carry one copy of the dTip100 element inserted in the GUS gene in the genome (see Table 2). Tip100 in pSUG2::Tip100 was introduced into the dTip1 plant by Agrobacterium-mediated transformation. All of the six independently obtained Hmr calli displayed a small but signi®cant number of GUS-positive blue cells when stained with X-Gluc (data not shown). No such blue cells could be detected in the Hmr calli obtained by introduction of the control vector pSUG2. The results further support the notion that Tip100 is an autonomous element and is able to act on the non-autonomous dTip100 element in the heterologous tobacco system. Footprints generated by excision of Tip100 and dTip100 One of the characteristics of plant transposable DNA elements, including those of the Ac/Ds family, is their ability to generate small sequence alterations at excision sites, which are referred to as footprints (Kunze et al. 1997). To obtain further evidence for transposition of the Tip100 element in tobacco, we determined the sequences of the Tip100 excision sites to examine whether footprints had been generated. The 450-bp fragments containing the Tip100 excision site were ampli®ed by PCR and cloned into plasmids. All 12 T0 plants with pSUG1::Tip100 that displayed the GUS-positive patches yielded ampli®ed fragments, while no ampli®cation products were detected in the transgenic plants with pSUG1::dTip100. Since these ampli®ed fragments are likely to contain di€erent footprints resulting from independent Tip100 excisions, we determined a total of 153 excision sequences consisting of at least 10 excision sequences from each of the 12 di€erent transgenic plants obtained (Table 1). Sixteen di€erent sequences were detected and ®fteen of them showed the characteristics of footprints. Of these, the two most frequently occurring sequences, Footprints 1 and 2, have a 1-bp deletion at either end of the original 8-bp TSD. Tip100 in pSUG1::Tip100 is ¯anked by the 8-bp TSDs originating from the CHS-D intron in the common morning glory (see Materials and methods), and these two footprints were previously observed following Tip100 excision from the ¯aked allele of the CHS-D gene (Habu et al. 1998). Footprint 16 contains one intact TSD followed by a 264-bp segment of the 5¢ end of Tip100 linked to the the other TSD which has a 2-bp deletion; the remaining 3.7 kb of Tip100 sequence including its 3¢ end has been deleted. A similar aberrant excision structure was found at the Arev3 allele in a revertant of the mutable af line of the common morning glory (Habu et al. 1998). To examine whether footprints were also generated at sites of Tip100-mediated excision of the dTip100 element

5¢ Flank cccctgcag CACGTATG

± 12

± ± 10

ACGTATGctgcag CACGTATGctgcag

±

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±

±

ATACGTGCTGC AGGTG CG (DTip100)

±

±

±

±

±

ATGctgcag

±

CACGTATGctgcag

±

CA

1

ACGTATGctgcag

±

CGTATGctgcag

±

tgcag

± ±

CAT

± ±

g ACGTATGctgcag

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ACGTATGctgcag

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cag

2 ±

6

GTG

3 ±

CACGTATGctgcag TATGctgcag

±

3

ACGTATGctgcag

4

12

2

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1

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2

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1 ±

1

5

20

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20

11

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1

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2 ±

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8

14

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1

1

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2 ±

2

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1

7

10

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1

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± 2

2

5

14

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8

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3

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8

11

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4

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19

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16

1

10

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3

7

Tip 2 Tip 3 Tip 4 Tip 5 Tip 6 Tip 7 Tip 8 Tip 9 Tip 10 Tip 11 Tip 12 (1) (1) (1) (1) (1) (3) (2) (1) (N. D.)(1) (1)

ACGTATGctgcag

3

Tip1 (3)

CACGTATGctgcag

3¢ Flank CACGTATGctgcag

GTG

Insert Tip100

Number of footprints found (copy number of T-DNA)c

153

2

9

2

1

2

2

1

1

1

3 1

3

11 2

36

76

Nd

1

2

2

1

1

2

1

1

1

2 1

2

6 1

8

12

Td

a Footprint sequences found in the 12 transgenic plants transformed with pSUG1::Tip100. The original sequence shown in the ®rst row represents the ¯anking sequences of Tip100 in pSUG1::Tip100 bTSDs and their ¯anking sequences are shown in bold upper case and in lower case letters, respectively. Deleted nucleotides are omitted, and newly generated sequences within the footprints are shown in the central column c The copy number of the NPTII gene in each transgenic plant is indicated in parentheses. N.D. indicates that the copy number was not determined. From each plant, at least 10 clones containing footprints were sequenced. The data represent the numbers of footprints found in each of the plants indicated d N and T denote the total numbers of clones and transgenic plants carrying the given footprint, respectively

cccctgcag CACGTAT 2 cccctgcag CACGTATG 3 cccctgcag 4 cccctgcag CACGTA 5 cccctgcag CACGTATG 6 c 7 cccctgcag CACGTA 8 cccctgcag CACGTA 9 cccctgcag CACGT 10 cccctgcag CACGTATG 11 cccctgcag CACGTA 12 cccctgcag CACG 13 cccctgcag CACGTAT 14 cccctgcag CACGTAT 15 cccctgcag CACGTATG 16 cccctgcag CACGTA Number of clones sequenced

1

Original

Footprinta Sequenceb

Table 1 Footprint sequences generated by Tip100 excision

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737 Fig. 3A±C GUS-positive patches observed in leaves of transgenic tobacco plants transformed with pSUG1::Tip100. A T0 plant. B, C T1 plants

Table 2 Footprint sequences generated by Tip100-mediated excision of dTip100 Footprinta

Sequence

Original

5¢ Flank cccctgcagCACGTATG

d1 cccctgcagCACGTAT d2 cccctgcagCACGTATG d3 cccctgcag Number of clones sequenced

Number of footprints found Insert dTip100

3¢ Flank CACGTATGctgcag CACGTATGctgcag ACGTATGctgcag CACGTATGctgcag

a

Footprint sequences found in the three Hmr calli derived from the transgenic plant dTip1 that were transformed with pSUG2::Tip100 , which carries one copy of the T-DNA (NPTII gene). The original sequence shown in the top row represents the ¯anking sequences of dTip100 in pSUG1::dTip100

in the Hmr calli derived from the dTip1 plant, we chose three independent GUS-positive calli transformed with pSUG2::Tip100. All of them gave rise to the 450-bp fragments containing dTip100 excision sites. We determined 11 footprints from these GUS-positive calli (Table 2) and found no signi®cant di€erence between the footprints generated by excision of dTip100 and those left behind on excision of Tip100. Therefore, we can conclude that excision of Tip100 generates footprints characteristic of transposable elements belonging to the Ac/Ds family. A 450-bp fragment could not be ampli®ed from the Hmr calli transformed with pSUG2.

Reintegration of Tip100 into the tobacco genome To examine whether the excised Tip100 elements were reintegrated into new sites in the genomes of the transgenic tobacco plants, we characterized the ¯anking sequences of Tip100 insertion sites that di€ered from the original site in the GUS gene. Since excision of Tip100 occurs at rather low frequencies, the reintegration of Tip100 was also expected to be a relatively rare event. We thus applied the IPCR procedure followed by the nested PCR ampli®cation technique with appropriate primers (Fig. 2), and were able to identify eight di€erent reintegration sites from four transgenic plants among the selfed T1 progeny derived from the GUS-positive T0 plants (Fig. 4). As expected, Tip100 generates 8-bp TSDs upon reintegration. Based on the results obtained in transgenic tobacco plants, we therefore conclude that Tip100 is an auton-

dTip1-1

dTip1-2

dTip1-3

2 1 ± 3

4 1 1 6

2 ± ± 2

Nb

Tb

8 2 1 11

3 2 1

b

N and T denote the total numbers of clones and Hmr transformants carrying the given footprint, respectively. The data are displayed as in Table 1

Fig. 4 Sequence analysis of Tip100 reinsertion in transgenic tobacco plants. Newly generated TSDs and their ¯anking sequences are shown in bold upper case and lower case letters, respectively

omous element coding for an active transposase of 808 amino acids. Apart from the autonomous Ac element (Baker et al. 1986), several other plant transposable elements of the Ac/Ds family, including Tam3 of snapdragon and Tag1 of Arabidopsis, are regarded as autonomous because they are capable of somatic transposition in transgenic tobacco plants (Haring et al. 1989, 1991; Martin et al. 1989; Frank et al. 1997). To our knowledge, Tip100 is the ®rst autonomous element to be characterized among the various transposable elements known in Ipomoea species (Hoshino et al. 2001). Tip100 and Modulator in the common morning glory Based on the observations that (1) lines of the common morning glory carrying the alleles af and yglm tend to show

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a striking similarity in both the timing and frequency of variegation in ¯owers and leaves, and (2) these variegated phenotypes are also generally heritable by their progeny, Kasahara (1956) postulated that there must be a factor (called Modulator) that acts on both af and yglm alleles, and that the timing and frequency of the variegation in ¯owers and leaves are determined by the state of the Modulator activity, which is generally heritable. Indeed, the CHS-D gene in the stable white-¯ower mutant carrying the mutable af allele without active Modulator is identical to that in mutable af lines displaying variegated ¯owers (Fig. 1), whereas the white-¯ower line carrying the stable a allele was found to have two copies of Tip100 integrated into the CHS-D gene (Habu et al. 1998). Although the simplest explanation for these observations is that Tip100 and Modulator represent a non-autonomous and an autonomous element, respectively, our present ®nding that Tip100 is an autonomous element is clearly incompatible with this assumption. The genome of the common morning glory contains about 100 copies of Tip100-related elements and the majority of them appear to be structurally very similar to Tip100 (N. Ishikawa, unpublished results), suggesting that a signi®cant number of autonomous Tip100 copies are present in the genome of I. purpurea. Similarly, it has been observed that most copies of Tam3 in the genome of snapdragon have highly conserved structures of nearly the same size (Kishima et al. 1999). Interestingly, both Tip100 and Tam3 carry single ORFs for active transposase that lack intron sequences (Hehl et al. 1991; Habu et al. 1998). A possible alternative hypothesis is that Modulator may be an element that a€ects the transposition activity of the autonomous Tip100 element. In the Ac/Ds family of elements, Stabilizer in snapdragon and the IAE loci in Arabidopsis have been reported to control the transposition activity of the autonomous elements, Tam3 and Ac, respectively (Carpenter et al. 1987; Jarvis et al. 1997). Recently, mutations in the DDM1 gene (encoding a SWI2/SNF2 chromatin-remodeling factor) that lead to hypomethylation of DNA have also been reported to result in activation of DNA transposable elements and developmental abnormalities in Arabidopsis (Kakutani et al. 1996; Miura et al. 2001; Singer et al. 2001). Since no apparent developmental abnormalities have been observed in lines of the common morning glory that display highly variegated ¯owers, it is, however, unlikely that Modulator is directly related to the DDM1 function. Acknowledgements We thank Y. Habu for comments at the initial stage of this study and A. Hoshino for invaluable discussion. This work is supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology in Japan. Y. J-H. received a Research Fellowship awarded by the Japan Society for the Promotion of Science for Young Scientists.

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